The present invention relates to an apparatus for counting and classifying particles of the type composed of a flow nozzle which hydrodynamically focuses the particles and which opens into a capillary nozzle that generates a thin stream having a circular cross section and containing particles which are aligned along an axis of the thin stream, and a jacket pipe which encloses the capillary nozzle and in which an entraining stream is formed for the thin particle stream.
For various purposes, it is necessary to count and record the size distribution of cells and particles with simultaneous classification according to certain cell characteristics. However, numerous problems have been encountered in procedures thus far devised for performing these operations.
One known process based on the Coulter principle provides for electronic measurement of the cell volume on the basis of changes in resistance of an electrolyte liquid during passage of the cells through an opening in a partition.
It has also been proposed to employ optical flow-through methods which entail fluorescence measurements with colored particles and differentiation according to the intensity of the fluorescence, or scattered light measurements in which a coherent light source is scattered at particles, or measurements of the absorption of the entire cell on object carriers. In all these optical processes the measuring volume is greater, however, than the cell or particle to be measured, respectively.
In measurement procedures according to the Coulter principle, the measured value depends on the geometry of the measuring opening in the partition and on the location of the path of travel of the particles through the measuring opening. No information other than cell volume can be obtained about the particles. Moreover, there exists a danger of the measuring opening becoming clogged and the maxium cell diameter is limited to 50% of the measuring opening. The result is a low counting rate which is still dependent on particle size.
Fluorescence measurements have the drawback that the measured value is dependent of the coloration processes, i.e., different measuring series cannot be directly compared with one another and fluorescence colorations of special cell characteristics can often not be produced at all. In the case of scattered light measurements it is necessary, in order to record a size distribution, to simultaneously effect measurements at various spatial angles. This has the result that only size distributions up to a maximum of about 10.mu. can be derived from scatter data. In the practice of both of these measuring methods, the particles are present in suspension and the optical quality of the suspension stream is also not optimally adapted to the index of refraction of the cells.
Absorption measurements have in the past been successful only when the measuring field is larger than the cell cross section. The cells are here applied to object carriers, and this results in low counting and analysis speeds since the object carrier must, inter alia, be moved mechanically.
To remedy these deficiencies, a more recently developed apparatus disclosed in German Offenlegungsschrift No. 2,543,310 and counterpart U.S. Pat. No. 4,110,043, performs the optical analysis of cells and particles in a fluid stream for the purpose of separating or enriching, respectively, particles and cells. The cells and particles are formed into a single file stream with the aid of hydrodynamic focusing and are individually aligned along the central flow axis. Oriented in such a manner, they leave the nozzle with the fluid common having a circular cross section. After leaving the hydrodynamic focusing nozzle, the stream of fluid divides after a short distance into individual droplets containing discrete particles or cells, respectively. Corresponding to the optical information obtained from the cells or particles during the passage through the measuring volume, which lies in the undisturbed region of the stream of fluid, they are deflected into different directions and sorted.
In one variation of this system, the measuring volume does not lie in the region of the upstream cuvette which is free of flow. It is here insignificant whether a further entraining fluid is used to compensate pressure losses. All experimental and commercial cytometric flow sorters involve embodiments in which the entrained stream of carrier fluid has a circular cross section.
However, the optical representation, or image, of the cells or particles disposed in such a stream of fluid is distorted in the plane perpendicular to the direction of flow by the carrier fluid which, because of its circular cross section, acts as a cylindrical lens. This makes planar representation of the center of the fluid column impossible. Moreover, the diffraction and reflection properties of the transition from the optically denser medium of the carrier fluid to the optically less dense entraining medium, which may be air, change the polarization properties of the light passing through these interfaces. With the recent increase of polarization optical examinations involving immunocompetent cells and image analysis in flow, this seems to be of particular significance for the future. An analysis of light scattering in a circular fluid steam is possible only in the direction parallel to the direction of the flow, whereas in a rectangular flow stream the correct imaging properties can be obtained and all solid angles within the forward lobe can be detected without distortion.